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. 2023 Nov 14;2(11):pgad394.
doi: 10.1093/pnasnexus/pgad394. eCollection 2023 Nov.

Readministration of high-dose adeno-associated virus gene therapy vectors enabled by ImmTOR nanoparticles combined with B cell-targeted agents

Petr O Ilyinskii  1 , Christopher Roy  1 , Alicia Michaud  1 , Gina Rizzo  1 , Teresa Capela  1 , Sheldon S Leung  1 , Takashi Kei Kishimoto  1
Affiliations

Readministration of high-dose adeno-associated virus gene therapy vectors enabled by ImmTOR nanoparticles combined with B cell-targeted agents

Petr O Ilyinskii et al. PNAS Nexus. .

Abstract

Tolerogenic ImmTOR nanoparticles encapsulating rapamycin have been demonstrated to mitigate immunogenicity of adeno-associated virus (AAV) gene therapy vectors, enhance levels of transgene expression, and enable redosing of AAV at moderate vector doses of 2 to 5E12 vg/kg. However, recent clinical trials have often pushed AAV vector doses 10-fold to 50-fold higher, with serious adverse events observed at the upper range. Here, we assessed combination therapy of ImmTOR with B cell-targeting drugs for the ability to increase the efficiency of redosing at high vector doses. The combination of ImmTOR with a monoclonal antibody against B cell activation factor (aBAFF) exhibited strong synergy leading to more than a 5-fold to 10-fold reduction of splenic mature B cells and plasmablasts while increasing the fraction of pre-/pro-B cells. In addition, this combination dramatically reduced anti-AAV IgM and IgG antibodies, thus enabling four successive AAV administrations at doses up to 5E12 vg/kg and at least two AAV doses at 5E13 vg/kg, with the transgene expression level in the latter case being equal to that observed in control animals receiving a single vector dose of 1E14 vg/kg. Similar synergistic effects were seen with a combination of ImmTOR and a Bruton's tyrosine kinase inhibitor, ibrutinib. These results suggest that ImmTOR could be combined with B cell-targeting agents to enable repeated vector administrations as a potential strategy to avoid toxicities associated with vector doses above 1E14 vg/kg.

Keywords: AAV redosing; adeno-associated virus; gene therapy; immune tolerance; immunogenicity.

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Figures

Fig. 1.
Fig. 1.
Low-dose ImmTOR inhibits anti-AAV8 IgG and enables increased SEAP expression after two AAV8-SEAP vector doses but has no effect at the third vector injection. A) Serum SEAP dynamics. Mice (5/group) were injected 3 times with AAV8-SEAP (5E11 vg/kg) on days 0, 93, and 176 either alone or coadministered with low-dose ImmTOR (50 µg). AAV readministration days are indicated by arrows. Fold increases in SEAP expression postsecond dose (d99–d188) vs. preredosing (d89) are shown (upper text above columns) and percent increase in SEAP expression vs. day 19 untreated AAV-SEAP group are shown for all the time-points in the AAV8-SEAP + ImmTOR-treated group (lower text above columns). SEAP levels prior to the second vector dose are indicated for the AAV8-SEAP group (lower-dotted line) and the AAV8-SEAP + ImmTOR group (upper-dotted line). B) AAV8 IgG dynamics. Anti-AAV8 IgG titers are shown for days 62 to 188. AAV readministration days are indicated by arrows. The number of mice that remain seronegative at day 188 is indicated.
Fig. 2.
Fig. 2.
IgM against AAV is the key factor determining lack of effective redosing in ImmTOR-treated mice with no detectable IgG to viral capsid. A–C) Passive immunization with anti-AAV8 IgG-negative serum prevents AAV transduction. Pooled sera from selected mice treated with ImmTOR + AAV8-SEAP were used to passively immunize naïve recipient mice 24 h prior to AAV8-SEAP administration. All individual serum samples used for the pool had low anti-AAV8 IgG (OD of 0.017 to 0.038 by AAV8 IgG ELISA) and no SEAP activity (described in Fig. S1B to D). Recipient mice were inoculated with 2, 20, or 200 µl of pooled sera, as indicated. A) SEAP dynamics. Relative SEAP levels vs. non-immunized control normalized to 100 are shown above each bar. B and C) De novo induction of anti-AAV8 IgM B) and IgG C) antibodies following AAV8-SEAP treatment in mice passively immunized with IgG-negative serum. D) Inhibition of in vitro AAV8-Luc transduction by untreated or heat-inactivated anti-AAV8 IgG-positive and IgG-negative serum. Pooled sera (from the studies described in Figs. 1 and S1) either possessing high levels of IgG against AAV8 or those determined to be IgG seronegative were maintained on ice or heat-inactivated at 63˚C for 10 min and then incubated with AAV8-Luc at various dilutions as indicated. The AAV8-Luc/sera admixture was then used to transduce Huh-7 cells in vitro, and luciferase activity was assessed 24 h later.
Fig. 3.
Fig. 3.
Ibrutinib combined with ImmTOR inhibits anti-AAV IgM responses in a dose-dependent fashion and enables successful vector readministration. A) Experimental scheme. Five groups of mice (6 each) were injected with 5E11 vg/kg AAV8-SEAP on days 0, 93, and 161. One group was treated with ImmTOR alone (100 µg) and three groups received ImmTOR combined with ibrutinib (17 daily injections at 20, 100, or 500 µg/day, starting 2 days before through 14 days after each AAV injection). B and C) Anti-AAV8 IgM B) and IgG C) dynamics after three AAV administrations. AAV8 dosing days are indicated by arrows. D) SEAP expression dynamics after two AAV8-SEAP redosings. AAV8 redosing days are indicated by arrows. Percent increase in SEAP activity at each time-point vs. untreated group as 100 is indicated (top line). Transgene boosting efficacy with each dose (ratio of post-redosing SEAP expression vs. that immediately before redosing) for each group is also shown (bottom line). Time-points with statistical difference between SEAP expression in groups treated with ImmTOR combined with ibrutinib and ImmTOR alone are indicated (**P < 0.01).
Fig. 4.
Fig. 4.
Quadruple AAV dosing in combination with ImmTOR and aBAFF. A) Experimental scheme. Six groups of mice (6 each) were injected with 5E11 vg/kg AAV8-SEAP 4 times on days 0, 32, 98, and 160. Group 1 received only the four AA8-SEAP injections. Groups 2 and 3 received low-dose ImmTOR (50 µg) concurrently with AAV, either without or with an 8-dose course of aBAFF (100 µg, days 0, 14, 32, 98, 112, 130, 160, and 174), respectively. Groups 4, 5, and 6 received a standard dose of ImmTOR (150 µg) concurrently with AAV without or with a 4-dose course (days 0, 32, 98, 160) or 8-dose course (days 0, 14, 32, 98, 112, 130, 160, and 174) of aBAFF (100 µg), respectively. B and C) Anti-AAV8 IgM B) and IgG C) dynamics after four AAV administrations. Days of AAV8 administration (days 0, 32, 98, and 160) are indicated by arrows; see panel A for details on dose regimen of other agents. Time-points with statistical difference between IgM levels in groups receiving 150 µg ImmTOR alone (third panel from the bottom) vs. those receiving the same ImmTOR dose combined with aBAFF (two bottom panels) are indicated in panel B (*P < 0.05, **P < 0.01). Numbers of mice (out of total) in each ImmTOR-treated group becoming IgG-positive by day 179 is shown in panel C. D and E) Serum SEAP expression dynamics after three AAV8-SEAP redosings in arms receiving low D) or standard E) dose of ImmTOR with or without aBAFF. AAV8 redosing days (days 32, 98, and 160) are indicated by arrows; see panel A for details on dose regimen of other agents. Relative levels of transgene expression for each experimental group vs. the untreated control (normalized to 100) is shown for each time-point as indicated in the top line above each bar. Transgene boosting efficacy of treatment following each round of AAV readministration is calculated as the ratio of postredosing SEAP expression vs. that immediately before redosing for each treatment group, as indicated in the bottom line above each bar. Time-points with statistical difference between SEAP expression in different groups are indicated (*P < 0.05, **P < 0.01).
Fig. 5.
Fig. 5.
ImmTOR and aBAFF synergize to decrease splenic B cells, plasmablasts, and elevate pro-/pre-B cells counteracting the activation by AAV antigen. A to C) Size of splenic fractions of CD19+ A), CD19+CD138+ B), and CD19+CD127+ C) cells at 1, 4 or 7 days after inoculation with AAV8 (5E11 vg/kg) alone or combined with ImmTOR (150 µg) and/or aBAFF (100 µg) as indicated. The size of each cell fraction vs. that of naïve mice (day 0, no treatment) as 100 (A, B) or 1.0 C) is shown. Three mice per each time-point in each group have been used.
Fig. 6.
Fig. 6.
ImmTOR and aBAFF enables readministration of high doses of AAV. A) Experimental scheme. Five groups of mice (8 each) were injected with either a single dose of 1E14 vg/kg AAV8-SEAP at day 0 (Group 1) or two doses of 5E13 vg/kg at days 0 and 56 (Groups 2–5). Animals receiving two injections of 5E13 vg/kg either received no further treatments (Group 2), monthly doses of ImmTOR (200 µg; Group 3), biweekly doses of aBAFF (100 µg; Group 4) through day 28 followed by monthly doses through day 112, or the combination of ImmTOR and aBAFF (group 5), as indicated. B and C) Anti-IgM B) and IgG C) dynamics (times of AAV8-SEAP dosing indicated by arrows). D) SEAP dynamics from day 19 to the end of the study (day 145). AAV redosing at day 56 is indicated by arrow. Day 19 SEAP level in Group 1 injected with 1E14 vg/kg AAV8 is shown by the dashed black line and that in Group 2 injected with 5E13 vg/kg AAV8 is shown by the dotted gray line. Transgene expression levels for each experimental group at each time-point vs. that in untreated group injected with 5E13 vg/kg AAV8 normalized to 100 are indicated (top line). Transgene boosting efficacy (ratio of postredosing SEAP expression vs. that immediately before redosing) for each group is also shown (bottom line). Statistical significance of differences between groups treated with ImmTOR vs. treated with ImmTOR and aBAFF combination in panels B to D is indicated as is the difference in SEAP levels between group dosed with 1E14 vg/kg AAV vs. group dosed twice with 5E13 vg/kg AAV and treated with ImmTOR and aBAFF (*P < 0.05, **P < 0.01, ***P < 0.01).
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